Apparatus and method for detecting canine cancer

ABSTRACT

It has been found that canine ECPKA protein secrets in a high level and an autoantibody against the canine ECPKA protein is formed in dogs with cancer. It is also found that human ECPKA does not selectively bind to a canine ECPKA autoantibody and cannot serve as a biomarker. In addition, canine ECPKA autoantibody detection can be used as a meaningful diagnosis tool for cancer in dogs only when quantitative measurement of such antibodies is adapted. When the measurement of canine ECPKA autoantibody is not conclusive, measuring CRP can provide supplemental data that can be used to improve the predictability of the canine ECPKA autoantibody measurement.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 371, of PCTInternational Application No. PCT/IB2017/056236, filed on Oct. 10, 2017,which claimed priority to U.S. Application 62/405,996, filed on Oct. 10,2016, the disclosures of which are hereby incorporated by referenceherein in their entirety.

SEQUENCE LISTING

A sequence listing has been submitted with this invention and isincorporated by reference herein, in its entirety.

FIELD OF THE INVENTION

The present invention relates to apparatus and methods for detectingcancer of a dog using canine PKA Cα protein as an antigen. The detectionmethod and apparatus utilize quantitative detection of antibodies thatcan be bound to a canine PKA Cα protein or its subunit with atherapeutically meaningful selectivity and specificity.

BACKGROUND

Human and dogs share many similarities genetically and immunologicallybut at the same time, exhibit many different immune responses andbiological responses as well. For example, humans are susceptible toAIDS virus but dogs are resistant to the virus. Also, dogs aresusceptible to canine distemper but humans are not.

There are many other examples where a potential bio-marker useful forhuman but not to dogs. For example, the S phase-specific proteinthymidine kinase 1 (TK1) has been known to be useful biomarker for humancancer as described in H. von Euler and S. Eriksson, Vet Comp Oncol.2011 March, 9(1):1-15. doi: 10.1111/j.1476-5829.2010.00238.x. Epub 2010Aug. 19. The expression of TK1 is tightly correlated to the fraction ofS phase cell and the level of proliferation. In normal cells, TK1activity is present only during the late G1 and early S phase but inmany tumor cells, TK1 activity is higher and remains throughout the Sand G2 phases. Excessive and uncontrolled cell proliferation is one ofhallmarks of cancer. Thus, the level of TK1 activity was examined todetermine its correlation with cancer and has proven useful fordiagnosis of and monitoring tumors such as solid tumor including breastcancer. Moreover, a major advantage in TK1 is that several monoclonaland polyclonal antibodies can be bound to TK1. The most specific andsensitive TK1 antibodies are produced against a 31-amino acid peptiderepresenting the C-terminus of TK1. The protein sequence homology in TK1is high between humans and dogs. H. von Euler and S. Eriksson. Theprimary amino acid sequences of canine and human TK1 are highlyhomologous from the N-terminal and for about 200 amino acids but theC-terminal regions differ.

In spite of the high homology in the protein sequence between human andcanine, the TK1 values in dogs having four different solid tumorsincluding breast cancer were found to be within a normal range. NakamuraN, Momoi Y, Watari T, Yoshino T, Tsujimoto H and Hasegawa A., Plasmathymidinekinase activity in dogs with lymphoma and leukemia, the Journalof Veterinary Medical Science 1997; 59: 957-960. Also, only three of 50tested dogs with a solid cancer showed an increased TK1 activities.Monitoring therapy in canine malignant lymphoma and leukemia with serumthymidine kinase 1 activity—evaluation of a new, fully automatednon-radiometric assay. International Journal of Oncology 2008; 34:505-510. Canine TK1 is not a useful biomarker for detecting cancer fordogs unlike human TK1. Moreover, the most sensitive and selectiveantibodies directed against human TK1 do not recognize canine TK1. H.von Euler and S. Eriksson. Similarly, antibodies directed canine TK1 atsimilar region cannot likely be recognized by human TK1 protein.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA), a serine/threonineprotein kinase mediating cAMP action in mammalian cells, is involved incontrolling various biological processes such as cell proliferation,differentiation, metabolism and apoptosis. Deregulation of PKA has beenlinked with the initiation and progression of cancer, and indeed itsoverexpression is frequently observed in different types of humancancer.

Hence, PKA has been suggested as a potential molecular target for adiagnostic biomarker in human cancer. Inactive PKA holoenzyme is atetramer composed of two catalytic and two regulatory subunits. Uponbinding with cAMP on regulatory subunits, the inactive PKA tetramer isdissociated into one dimer of regulatory subunits and two monomers ofactive catalytic subunits, which then phosphorylate various targetproteins in both nucleus and cytoplasm. Four isoforms of regulatorysubunit, RIα, RIβ, RIIα and RIIβ, have been identified throughbiochemical studies and outcomes of PKA signaling activation has beenshown to depend on types of regulatory subunit isoforms in cells. Theexpression of RII isoforms is preferentially observed in normal tissuesand inhibits the growth of cells, whereas the expression of RI isoforms(RIα/PKA-I) stimulates cell proliferation.

In particular, overexpression of the RIα/PKA-I is highly correlated withcancer progression, multidrug resistance and various types of cancerpatients with a poor prognosis. Interestingly, recent studies show thatPKA is expressed mostly intracellularly in normal cells, whereas anextracellular form of the PKA (ECPKA) is secreted from numerous types ofcancer cell lines including prostate, bladder, breast, colon carcinomaand lung adenocarcinoma. In addition, ECPKA was highly detected in serafrom human patients with various types of cancer but not in sera fromhealthy volunteers. Furthermore, it was also shown that the serum levelof ECPKA decreases after surgical resection of tumors in human melanomapatients. See U.S. Pat. No. 7,838,305B2, which report compositions andmethods for the detection of anti-ECPKA autoantibodies using human ECPKAas an antigen. These observations raise the possibility that human ECPKAand its autoantibodies in serum could be a valuable diagnostic for humancancer.

Canine ECPKA has different amino acid sequence with human ECPKA. Theamino acid sequences are shown in FIG. 1. The different sequences arespread throughout the chain including C-terminus. It is not knownwhether canine ECPKA present in dogs with cancer and, in particular,whether the level of the canine ECPKA present in dogs can be correlatedwith the presence of cancer. Moreover, it is not known whether dogs haveimmune responses to canine ECPKA creating autoantibodies or even if so,whether such immune response strong enough to be useful for a biomarkerto detect canine cancer. It is also not known whether human ECPKA can beused as an antigen for a diagnostic purpose to detect canine cancer.Typically, a level of antigen such as ECPKA in a plasma may decay overtime and it may be difficult to adopt a quantitative analysis of anantigen for a diagnostic measurement. The detection of a level ofantigen may not be a useful tool for diagnosis or detection with acertain probability because the retention time of the sample beforesubjecting the test would affect the result. However, a level of anautoantibody is much more free of such decay and can be useful as abiomarker especially when a quantitative control is required.

Through extensive research and studies over different breeds and ages ofdogs with various types of cancer, it has been found that dogs showdistinctive canine ECPKA and autoantibody activities, which can be usedto detect the presence of cancer in a dog and there is no or very littlecorrelation between human ECPKA and canine cancer detection. Due to theunique distinctiveness, a quantitative detection of the antibodies forcanine ECPKA, rather than a simple qualitative detection, is desirableand a device enabling such quantitative detection is developed. Inparticular, the quantitative detection is developed to be adopted indigitized data process and a device assisting such digitized process isalso developed. The quantitative detection device can be used fordetecting other proteins or virus agents as described herein.

SUMMARY OF INVENTION

One embodiment provides a method for determining presence of cancer in adog by preparing a serum sample from the dog and detecting an amount ofan antibody in the serum sample using a purified recombinant canine PKACα protein as an antigen wherein the presence of cancer in the dog isdetermined when the amount of the canine PKA Cα antibody is above apredetermined level. Depending on the detected amount of the canine PKACα antibody, the possibility of the cancer presence can be determined.The purified recombinant canine PKA Cα protein is synthesized by using aprimer with a sequence SEQ ID NO. 1 (AAT CCA TGG GCA ACG CCG CCG CCA AGAAGG GCA G) and SEQ ID NO. 2 (GCC GTC GAC GAA CTC ACA AAA CTC CTT GCC ACACTT C). The purified recombinant canine PKA Cα protein is prepared byusing amplified cDNA fragments of canine PKA Cα and a bacterialexpression vector with T7 promoter and terminator primers havingsequences respective SEQ ID NO. 3 (AAT ACG ACT CAC TAT AGG) and SEQ IDNO. 4 (GCT AGT TAT TGC TCA GCG G). The resulting purified recombinantcanine PKA Cα protein has an amino acid sequence comprising SEQ ID NO. 5and a nucleotide sequence comprising SEQ ID NO. 6.

The predetermined level can be determined in consideration of variousfactors such as presence of other medical conditions such as liverdisease or inflammatory conditions. For example, dogs with a liverdisease may have exhibit a higher level of canine ECPKA autoantibodies.Thus, the method may have a preset instruction for dogs without a liverdisease. The predetermined level may be 3.5 μg/ml, preferably 4 μg/ml,more preferably 4.5 μg/ml, even more preferably 5 μg/ml.

It is also found that a high level of the amount of CRP is determined indogs with cancer. Accordingly, determining the amount of CRP can be usedin conjunction with the ECKPA or ECPCKA autoantibody detection todetermine the presence of cancer in a dog. Because CRP may presence muchmore predominantly in the serum than the ECPKA or ECPKA autoantibodies,a higher level of the CRP amount can be set to be a threshold level.Moreover, the CRP amount may be measured after further diluting theserum sample using a buffer solution. The dilution factor may be 100times. The predetermined CRP level may be about 80 μg, preferably 100 μgor even more preferably 120 μg.

Another embodiment provides a method for determining presence of cancerin a dog preparing a serum sample from the dog detecting an amount of acanine PKA Cα antibody in the serum sample using a purified recombinantcanine PKA Cα protein as an antigen and determining an amount of CRP inthe serum sample, wherein the presence of cancer in the dog isdetermined when the amounts of the canine PKA Cα antibody and CRP arerespectively above a predetermined antibody level and a predeterminedCRP level and wherein the amount of CRP is measured with an assistanceof a diluting buffer solution. The dilution buffer solution dilutes theserum sample by a factor of between about 50 and about 150.

Another embodiment provides a device for quantitatively detecting anantibody for canine PKA Cα protein a solid phase having an immobilizedpurified recombinant canine PKA Cα protein wherein the recombinantcanine PKA Cα protein has an amino acid sequence comprising SEQ ID NO.5, wherein the device is capable of detecting an amount of the antibodyfor canine PKA Cα protein using the recombinant canine PKA Cα protein.When the detected amount is above a predetermined level, cancer maypresence in a high possibility. The device for quantitatively detectingan antibody for canine PKA Cα protein may include a ligand capable ofbinding canine lgG wherein the ligand including a portion that is activeto either UV or visible light. The device may include a housing with asample reception portion, detection window wherein the solid phase isplaced within the housing and is capable of moving a sample receivedfrom the sample reception potion through a portion of the solid phaseexposed by the detection window.

The device for quantitatively detecting an antibody for canine PKA Cαprotein may be able to determine at least two levels of the amount ofthe detected antibody. The device may have a reference line which can beused as a reference quantitative line. The reference line shows asimilar color result regardless of the sample and allows quantitativedetermination of the amount of the antibody detected. The device ispreferably capable of determining multiple levels of the amount of thedetected antibody and gaps between two adjacent levels of the multiplelevel is less than about 5 μg/ml or less, preferably 3 μg/ml or less,even more preferably 1 μg/ml or less.

The device for quantitatively detecting a canine ECPKA autoantibody mayinclude one or more data input slots, which allows simultaneouscollection of patient data while the result of the device is taken. Forexample, the result of the device can be read using a reader by taking aphoto of the device and the test result can be read and converted to adigital data. The conversion process can convert the image of the solidphase shown in the detection window and also at the same time, convertthe data shown in the data input slots into a digital data for process.Such converted data can be sent to a remote server through acommunication device or module that may be incorporated in the testreader. The transmitted data can be compiled and organized foranalyzing. The analyzed data can be sent back to the test performer viathe test reader or other electronic means such as email, message, etc.The test reader may use a smart phone to take a photo and transmit thephoto to the remote server.

Another embodiment provides am apparatus for detecting a protein orviral agent in a mammal may have a housing with an outer surface and aninner space, a control panel on the outer surface, a camera located in away to be able to take a picture of a protein or viral detection samplein the inner space, a sample holder formed in the inner space whereinthe sample can be placed on the sample holder, a light source capable ofilluminating the inner space; a light dissipating device configured todissipate light from the light source allowing indirect illumination onthe sample, a communication module, wherein the light source and thelight dissipating device are configured to indirect illumination of thelight from the light source on the sample holder and the communicationmodule is configured to send the picture taken by the camera to a remoteserver.

The sample holder is configured to hold a lateral flow kit or other bioassay kit. The sample holder allows to locate the sample within a rangeof the camera so that the camera can take a picture of the sample. Thecamera and the communication module may be incorporated into thehousing. Alternatively, the camera and communication are part of a smartphone where the smart phone is placed on the housing to take a pictureof the sample and send the picture to a remote server. The smart phone'sflash light can be used the light source. The light dissipating devicemay be a semitransparent plate and is movable to be located directlybeneath of the light source of the smart phone.

The apparatus detecting a protein or viral agent in a mammal may includea removable smart phone adopter having an camera opening wherein thelight dissipating device is incorporated into the smart phone adopter ina way to cover the light source in the smart phone and the cameraopening is aligned to the camera of the smart phone wherein the housingfurther comprising a receiving area for the removable smart phoneadopter is placed wherein the receiving area has one or more opening forthe camera and the light source. The smart phone adopter may beincorporated into the housing.

The light source may be capable of illuminating light with apredetermined wave length such as UV light or mono wave light. Thecommunication module may use a short distance communication protocolsuch as WiFi, WiMx, Bluetooth, ZigBee, Z-Wave or other short distancecommunication protocol.

Another embodiment provides a method of detecting and monitoring aninfectious disease in a mammal includes testing the infectious diseasein a mammal using a kit having a viral detection agent, determining theresult of testing using a kit reader with camera and communicationcapabilities, sending the test result obtained by the kit reader to aremote server; and alerting a predetermined entity by an electricalcommunication method is provided. The method may utilize a kit thatprovides a UV active result and the camera is capable of detecting theUS active result.

The kit reader may include a housing having an outer surface and aninner space, a control panel on the outer surface, a sample holderformed in the inner space wherein the sample can be placed on the sampleholder, a light source capable of illuminating the inner space; and alight dissipating device configured to dissipate light from the lightsource, wherein the camera located in a way to be able to take a pictureof a protein or viral detection sample in the inner space, the lightsource and the light dissipating device are configured to indirectillumination of the light from the light source on the sample holder andthe communication module is configured to send the picture taken by thecamera to a remote server.

The test reader or kit reader may have GPS capability and is able toidentify the location of the test.

Another embodiment provides a lateral flow kit for quantitativelydetecting an antibody of canine ECPKA in blood of a dog, having a firstsolid phase having an immobilized purified recombinant canine PKA Cαprotein wherein the recombinant canine PKA Cα protein has an amino acidsequence comprising SEQ ID NO. 005, a conjugate pad comprising aconjugated coloring agent with an optical density wherein the conjugatedcoloring agent is conjugated with a first binding protein, and a secondsolid phase comprising a second binding protein, wherein the conjugatedcoloring agent is configured to provide a first color intensity in thefirst solid phase and a second color intensity in the second solid phaseand wherein the first color intensity quantitatively depends on theconcentration of the antibody in the blood of the dog and the secondcolor intensity is independent of the concentration of the antibody.

Because the concentration of the antibody is measured quantitativelyusing simple the lateral flow kit, the coloring agent may be appliedusing a pressurizing device and the optical density of the coloringagent may preferably be used in a high level. The optical density of theconjugated coloring agent may be between 5-30, preferably 8-25, morepreferably 10-20.

The first color intensity provides the concentration of the antibody bycomparing the first color intensity with a set of correlating databetween the first color intensity and the concentration of the antibody.The concentration can be extrapolated using the set of the data, whichcan be expressed into a correlation equation, preferably a linearequation.

The first binding protein may be selected from a group of streptavidin,biotin, protein A, anti-canine IgG Rat, anti-canine IgG Rabbit,anti-canine IgG goat, anti-canine IgG sheep and a protein capable ofbinding to the antibody. The second binding protein is selected from agroup of streptavidin, biotin, protein A, anti-rat IgG, anti-Rabbit IgG,anti-goat IgG, anti-sheep IgG and a protein capable of binding to IgG.Selection of the first and second binding proteins needs to becomplimentary.

The coloring agent may be selected from a group of gold, latex, gfp,fitc, and UV active conjugating agent. Different sizes of gold nanoparticles may be used.

The lateral flow kit may further have a filter phase to filter bloodcells, which allows directly applying a blood sample rather than serum.

Another embodiment provides a method of determining a concentration ofan antibody of canine ECPKA in blood of a dog where the method includessteps of (a) preparing a test sample from the blood of the dog for alateral flow kit comprising a test line; (b) applying the test sample tothe lateral flow kit; (c) allowing the lateral flow kit to develop; (d)obtaining a digital information by taking a digital picture of thedeveloped lateral flow kit including the test line; and (e) obtainingthe concentration of the antibody wherein the concentration isdetermined by comparing the digital information of the test line with aset of data correlating a digital value obtained from the digitalpicture with a concentration of the antibody.

The lateral flow kit used in this embodiment may be the lateral flow kitdescribed herein. The method may also include a step determining alikelihood that the dog has a cancer using extrapolation data set. Theextrapolating data may be expressed with a linear equation with an R²value higher than 0.9.

The method may involve sending the digital information to an externalserver via a wireless communication. The digital information may beobtained using a reader box comprising a wireless communication module,a camera module, a light source, and a slot designed to accommodate thelateral flow kit. The wireless communication module operates based on ashort range wireless communication protocol instead of a mobile networkallowing the method being conducted without any mobile phone but only ashort-range communication such as Wi-Fi.

The concentration of the antibody may be determined by the method in alevel that is an order of less than about 5 μg or less.

Another embodiment provides a method of determining a concentration ofan antibody of ECPKA in blood of a mammal, including (a) preparing atest sample from the blood of the mammal for a lateral flow kitcomprising a test line; (b) applying the test sample to the lateral flowkit; (c) allowing the lateral flow kit to develop; (d) obtaining adigital information by taking a digital picture of the developed lateralflow kit including the test line; and (e) obtaining the concentration ofthe antibody wherein the concentration is determined by comparing thedigital information of the test line with a set of data correlating adigital value obtained from the digital picture with a concentration ofthe antibody, wherein the lateral flow kit includes a first solid phasehaving an immobilized purified recombinant mammal PKA Cα protein whereinthe recombinant mammal PKA Cα protein has an amino acid sequence, aconjugate pad comprising a conjugated coloring agent with an opticaldensity of 5 or higher wherein the conjugated coloring agent isconjugated with a first binding protein, and a second solid phasecomprising a second binding protein. The conjugated coloring agent isconfigured to provide a first color intensity in the first solid phaseand a second color intensity in the second solid phase. The first colorintensity quantitatively depends on the concentration of the antibody inthe blood of the dog and the second color intensity is independent ofthe concentration of the antibody.

Various embodiments disclosed here may be combined as whole orselectively.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows comparison between amino acid sequences of a subunit ofhuman ECPKA and a subunit of canine ECPKA.

FIG. 2 shows comparison between mRNA sequences of a subunit of humanECPKA and a subunit of canine ECPKA.

FIG. 3 illustrates canine ECPKA autoantibody measurements of normaldogs, dogs with benign tumors, dogs with tumors and dogs that have beensurgically treated for cancer.

FIG. 4 shows a illustrative receiver operating characteristic graphwhere A is the area under the ROC curve.

FIG. 5 illustrates an ROC curve of a cancer detection method accordingto an embodiment of the invention.

FIG. 6 illustrates relationships between CRP level in a plasma andvarious tested subjects.

FIG. 7 illustrates relationships between CRP level in a plasma andvarious tested subjects.

FIG. 8 illustrates correlation between CRP measurements and canine ECPKAautoantibodies level.

FIG. 9 illustrates correlations between human ECPKA and canine ECPKA indetecting cancers of dogs.

FIG. 10 illustrates correlations between color intensity obtained usingone embodiment lateral flow kit and the concentration of the antibody ina sample wherein the correlation is approximately leaner with R² valuehigher than 0.9.

FIG. 11 illustrate the top view of a reader according to one embodiment.

FIG. 12 illustrate the bottom plate inside of a reader according to oneembodiment.

FIG. 13 illustrate a side view of a reader according to one embodiment.

FIG. 14 illustrate a side view of a reader according to one embodiment.

FIG. 15 illustrate an see-through view of a reader according to oneembodiment.

FIG. 16 illustrate an see-through view of a reader according to oneembodiment.

FIG. 17 illustrates test results of a kit according to one embodiment.

FIG. 18 illustrates an ROC curve of test results of a kit according toone embodiment.

FIG. 19 summarizes test results of a kit according to one embodiment.

DETAILED DESCRIPTION OF THE INVENTION

Through extensive researches, it has been found that canine ECPKAprotein secrets in a high level and an autoantibody against the canineECPKA protein is formed in dogs with cancers. It is also found thathuman ECPKA does not selectively bind to a canine ECPKA autoantibody andcannot serve as a biomarker. In addition, canine ECPKA autoantibodydetection can be used as a meaningful diagnosis tool for cancer in dogsonly when quantitative measurement of such antibodies is adapted. Whenthe measurement of canine ECPKA autoantibody is not conclusive,measuring CRP can provide supplemental data that can be used to improvethe predictability of the canine ECPKA autoantibody measurement asdescribed further below.

FIG. 1 is alignment of amino acid sequences of PKA Cα from human(NP_002721.1), dog (NP_001003032.1) and cat (XP_006928552.1). Dot boxesindicates amino acid residues showing difference between human and dog.While the amino acid sequences of human and dog share highlysimilarities but there are four different sequences are spread over theentire sequences including the c-terminus.

FIG. 2 compares mRNA sequences encoding PKA Cα from human (NP_002721.1),dog (NP_001003032.1) and cat (XP_006928552.1). There are a large numberof differences in between mRNA sequences of human and dog.

FIG. 3 illustrates the test results of ECPKA autoantibody measurementsin various test subjects: dogs (“Cancers” or “Cancer”) diagnosed ashaving cancer, dogs (“Benign tumor”) with benign tumor, dogs (“Control”or “Non tumor disease”) with no tumor disease, and dogs (“Tx” or“Treatment”). As shown in FIG. 3, the test subjects with cancers showmuch a higher level of ECPKA autoantibody measurement comparing to theother test subjects. Dogs that had tumor diseases but were treatedthrough surgery show low levels of ECPKA autoantibody measurements.Table 1 summarizes total numbers of the test subjects and test results.For categorizing the test results, the positive results were countedwhen the ECPKA autoantibody is detected 41 unit and higher, and thenegative results were determined when the level of the ECPKAautoantibody detection is less than 41 unit.

TABLE 1 Cancer Control Benign Tumor Treatment Positive 81 25 2 0Negative 1 160 23 19 Total 93 185 25 19

Table 2 summarizes the sensitivities, specificity, positive predictivevalue, and negative predictive value for the canine ECPKA autoantibodydetection as a cancer diagnosis method.

TABLE 2 Selectivity  87% Positive Predictive Value  75% Specificity88.4% Negative Predictive Value 94.4%

There are many ways to verify or evaluate accuracy of a particular testmethod. Among those, sensitivity and specificity are commonly used. Ingeneral, the sensitivity and specificity means how good a method is indistinguishing between the targeted result and untargeted result. Forexample, in our case, the sensitivity means how well cancers in dogs canbe found by using the detection of the autoantibody of canine ECPKA. Onthe other hand, the specificity relates to how well the method coulddistinguish dogs with cancer from dogs without cancer. In addition tothe sensitivity and specificity, Receiver Operating Characteristics(ROC) curve is often used to determine usefulness and cut-off value of amethod. The ROC curve is drawn using the rate of false positive in xaxis value and the rate of true positive in y axis value. Whether aparticular test method is accurate or not can be measured by using theROC curve. In the below illustrative ROC curve, in which the positiveproportion is plotted against the false positive proportion for variouspossible settings of the decision criterion, the area under the ROCcurve (AUC) of 1 means that the test method is perfect and accurate butif the AUC is 0.5, the test method is useless and inaccurate. As thecurve is closer to the upper left corner, the test method is moreaccurate and more useful. Typically, it is treated that a test method isnot informative when the AUC is 0.5, is not so accurate when the AUC isbetween 0.5 and 0.7, is accurate when the AUC is between 0.7 and 0.9,and is very accurate when the AUC is 0.9 and 1. Accordingly, the ROCcurve and its AUC value are a good tool to determine and evaluate a newtest method. See Using the Receive Operating Characteristic (ROC) Curveto Measure Sensitivity and Specificity, Korean J. Fam. Med. Vol. 30, No.11, November 2009, 30:841-842.

FIG. 5 shows a ROC curve of the canine ECKPA autoantibody measurementfor detecting cancer of dogs. The AUC value is 0.9061, which indicatesthat the canine ECKPA autoantibody measurement of one embodiment of thepresent invention is a highly effective and accurate diagnostic tool.

FIG. 6 shows measurements of C-reactive protein (CRP) in various testsubjects: dogs with cancer, dogs with benign tumors, dogs with no tumordiseases, dogs with non-cancer diseases. FIG. 7 shows measurements ofCRP in dogs with cancer (“Cancer”), dogs with false native results inthe canine ECKPA autoantibody measurement (“FN”), dogs with benigntumors (“BT”), dogs without cancer (“Negative”) and dogs with falsepositive results in the canine ECKPA autoantibody measurement (“FP”).

FIG. 8 plots the CRP measurement results against the canine ECKPAautoantibody measurement results of the various test subject groups.Table 3 show negative predictive values and positive predictive valueswhen the CRP and the ECKPA autoantibody measurements are used together.The positive predictive values increase to 91% and 100% from the overallpositive predictive value of 73.6% in the areas of C and E, and thenegative predictive value improves to 97.6% in the area of C from theoverall negative predictive value of 93.4%. Thus, measurement of CRP canimprove the accuracy of the cancer diagnosis using the canine ECKPAautoantibody measurement.

TABLE 3 Benign Area NPV (%) PPV (%) Cancer Tumor Non-tumor A 62 38 8 4 9B 97.6 2.4 4 19 138 C 9 91 20 0 2 D 50 50 25 2 23 E 0 100 13 0 0 F 11 8916 0 2

FIG. 9 is a chart that the test results of dogs based on human ECPKA isplotted against the test results of dog based on canine ECPKA. When ther² value is 1, the test results closely correlate to each other andhuman ECPKA or its autoantibodies can be used to detect cancer in dogs.However, as shown in Fig. x, the r² value is 0.15, suggesting there isnot much correlation between human ECPKA and Dog ECPKA and human ECPKAor its autoantibodies is not a good biomaker to detect cancers of dogs.

The table 4 summarizes various cancer detected in the test subjects. Itis also found that the cancer detection method of one embodiment of thepresent invention can be used regardless of the cancer type, which makesthe detection method unique and highly useful.

TABLE 4 Tumor type (Number) Diagnosis (Number) Carcinoma (57) Mammarygland Carcinoma (14) TCC (11) Hepatic carcinoma (9) Adenocarcinoma (5)SCC (5) Other carcinoma (13) Hematopoietic cancer (2) Lymphoma (14) Mastcell tumor (5) Leukemia (1) Sarcoma (16) Hemangiosarcoma (5) Melanoma(4) Soft tissue sarcoma (4) Other sarcoma (3) Benign (25) Adenoma (9)Lipoma (5) Histiocytoma (2) Benign mixed tumor (2) Other benign tumors(7)

The lateral flow kit structure may follow typical lateral flow kitstructures. However, the coloring agent and proteins used are speciallydesigned to detect the antibody of the canine ECPKA in a quantitativeway. The kit has an application place where a test sample is applied.The test sample is typically prepared from blood of a test subject.Serum obtained from the blood is applied to the application place. Theserum sample flows along the test strip and passes through a conjugatepad where a conjugated coloring agent is placed. The coloring agent isapplied with pressure and baked in oven. Typical optical density used inlateral flow kits is around 2-3. However, a much higher optical densityis used to obtain better correlation between the digitized test resultof the expression and the concentration data. Various coloring agent canbe used. Gold and latex are typical coloring agent. For UV activecoloring agents include gfp, fitc, and UV active conjugating agents. Thecolor agent exhibits different color intensity. The inherent intensityaffects the digitization. FIG. 10 shows an example correlation betweenthe color intensity received after testing using a lateral flow kitaccording to one embodiment and the concentration of the antibody.

The antibody of the canine ECPKA and other antibodies in the sample willbind to the conjugated first binding protein, effectively coating theantibodies with the coloring agent. The first binding protein may beselected from a group of streptavidin, biotin, protein A, anti-canineIgG Rat, anti-canine IgG Rabbit, anti-canine IgG goat, anti-canine IgGsheep and other protein capable of binding to the antibody. When thesample passes through the first solid phase of the kit, which containsan immobilized purified recombinant canine PKA Cα protein, the antibodyof the canine ECPKA binds to the immobilized purified recombinant caninePKA Cα protein. After the complete development of the kit, only theantibody of the canine ECPKA stays in the first solid phase, exhibitinga color intensity, which can be used to find the correspondingconcentration of the antibody. The expressed test result is digitized bytake a digital camera and the digitized information is compared with thecorrelation data to determine the actual concentration. The digitizedinformation uses color intensity to obtain a digital expression value.The embodiment shown in FIG. 10 provides a linear relationship betweenthe digitized expression value and the concentration. Thus, it allowsextrapolation of a concentration for which matching data does not existin the data set. For easier extrapolation, the color intensity of thecoloring agent for various concentrations of the antibody is desirableto provide a linear relationship.

Types of camera, color temperature setting, distance between the sampleand camera, light source and exposure setting would affect the digitizedvalue of the color intensity. Thus, it is desirable to have thedigitization of the expressed color intensity is desirably done in aconstant condition. FIGS. 11-16 shows a reader box 100, which has ahousing 200. A camera module 300, monitoring and controlling unit 400and bar code scanner 500 are provided on the top surface of the housing.Inside the reader, there is a slot 600 where the kit is placed throughthe opening 900. With the internal lighting system 1000 such as LED, thelighting inside of the reader is controlled to optimize to provide aconstant condition for the digitization. When the digital camera moduletakes a picture of the kit on the slot, the digital image is transferredto a server via the wireless communication module 100. The wirelesscommunication module uses a short distance communication protocol,allowing the reader to connect to a local internet portal such as WI-FIhot spot. The wavelength of the lighting source can be adjusted forvarious coloring agents such as UV active coloring agents. Because thereader has own communication module, it can operate independent of amobile network.

FIGS. 17-19 shows test results of a kit according to an embodiment wherethe kit shows sensitivity of 84.7% and specificity of 84.4%.

EXAMPLES Cloning of Canine Cyclic AMP-Dependent Protein Kinase CatalyticSubunit Cα (PKA Cα)

Total RNAs were isolated from canine adipose tissue homogenized inTrizol reagent (Invitrogen) using RNeasy columns (Qiagen). 1 μg of totalRNA was then reverse transcribed to cDNA with oligo (dT) primers usingImprom-II™ Reverse Transcription System (Promega) according tomanufacturer's instructions. The canine PKA Cα cDNA was amplified bypolymerase chain reaction (PCR) using exTaq polymerase (Takara) and thecanine PKA Cα primers containing restriction enzyme recognition sites,NcoI and XhoI. The sequences of primers are as follows: forward, AAT CCATGG GCA ACG CCG CCG CCA AGA AGG GCA G and reverse, GCC GTC GAC GAA CTCACA AAA CTC CTT GCC ACA CTT C.

The amplified cDNA fragments of canine PKA Cα was then digested withrestriction enzymes, NcoI and XhoI (Takara), inserted into pET-22b(+)plasmid (Novagen), a bacterial expression vector and sequenced with T7promoter and terminator primers (T7 promoter primer, AAT ACG ACT CAC TATAGG and T7 terminator primer, GCT AGT TAT TGC TCA GCG G).

Purification of Recombinant Canine PKA Cα

pET-22b(+) plasmid encoding canine PKA Cα tagged with six histidineresidues (6×-His Epitope) at the C-terminus was introduced intoEscherichia coli strain, BL21(DE3) and the expression of canine PKA Cαwas induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) atroom temperature for overnight. Cells were harvested, resuspended in 50mM Tris.HCl (pH 7.4) containing 0.2M NaCl and sonicated. Recombinantcanine PKA Cα was then purified with two sequential immobilized metalaffinity chromatography using IDA Excellose resin (Bioprogen) followedby ion exchange chromatography using SP Sepharose resin (GE healthcare).The eluted recombinant protein was dialyzed and stored at aconcentration of 1 mg/ml in 50 mM Tris.HCl (pH 7.4) supplemented with0.15M NaCl and 1% sucrose at −80° C. until further use.

Western Blotting

50 ng of purified recombinant canine PKA Cα and human PKA Cα, a positivecontrol, proteins were separated on 10% SDS PAGE gel, transferred ontoPVDF membrane and sequentially probed with rabbit polyclonal anti-PKA Cαantibody (Abcam) and goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (Bethyl Laboratories) followed byimmunodetection with enhanced chemiluminescence (Pierce).

Enzyme-Linked Immunosorbent Assay (ELISA) Assay

The presence and level of extracellular PKA Cα in canine serum wasassessed with anti-canine PKA Cα ELISA kit (Genorise) following themanufacturer's instructions. Briefly, 100 μl of 4-fold diluted canineserum samples in reagent diluent was added to the 96 well ELISA platesprecoated with anti-canine PKA Cα antibodies and incubated for 1 hr atroom temperature. The plates were further incubated with canine PKA Cαdetection antibodies for 1 hr at room temperature followed by incubationwith HRP conjugate for 20 min at room temperature. The plates were thendeveloped with substrate solution for 10 min at room temperature and thereaction was stopped with 50 μl stop solution. The absorbance wasdetermined at 450 nm with a scanning multi-well spectrophotometer(Molecular Device).

Autoantibodies against extracellular PKA Cα in canine serum weremeasured using solid phase ELISA method. Briefly, 96 well polystyreneELISA strip plates (Santa Cruz) were coated with 100 μl of recombinantcanine PKA Cα diluted at 1 μg/ml in carbonate coating buffer (pH 9.6)(Sigma) for overnight at room temperature, washed once with PBScontaining 0.1% Tween 20 (pH 7.4), blocked with 1% bovine serum albumin(BSA) in PBS for 2 hrs at room temperature and washed twice with washingbuffer (50 mM sodium citrate supplemented with 0.15M NaCl and 0.1% Tween20 (pH 5.2)). The plates were then incubated with 100 μl of canine serumsamples diluted at 1:500 in sample dilution buffer (PBS containing 0.25%BSA and 0.05% Tween 20 (pH 7.4)) for 1 hr at room temperature, washedfour times with washing buffer, further incubated with 100 μl of goatanti-canine IgG antibody (Abcam) conjugated with HRP diluted at 1:20,000in sample dilution buffer for 1 hr at room temperature, washed fivetimes with washing buffer, and developed with 100 μl of3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate solution(Thermo-Fisher) for 15 min at room temperature. The reaction was thenstopped with 50 μl of 2N H₂SO₄ solution and the absorbance was measuredat 450 nm using a scanning multi-well spectrophotometer.

Detection of Extracellular PKA Cα in Sera from Dogs with MalignantTumors

It has been shown that extracellular PKA produced by cancer cells ismarkedly increased in the sera of cancer patients and elicits thegeneration of autoantibodies against this it in those patients. Inaddition, it was reported that the titer of autoantibodies for PKA inserum is significantly correlated with the presence of cancers ofvarious cell types. Hence, the autoantibody against PKA is considered asa novel potential biomarker for diagnosis of cancers in human. However,the presence of PKA autoantibody and its correlation with cancer havenever been determined in mammals other than human.

Detection of Autoantibodies against Extracellular PKA Cα in Sera fromDogs with Malignant Tumors

To determine whether the titer of PKA Cα autoantibodies is positivelycorrelated with cancer of various cell types in dogs as shown in humans,first, we cloned canine PKA Cα gene from canine adipose tissues,bacterially expressed and purified the recombinant protein tagged with6×-His Epitope (Figure). The presence and titer of PKA Cα autoantibodieswere then assessed with ELISA assay using the purified canine PKA Cαprotein as an antigen.

Preparation of a Lateral Flow Kit

ECPKA (0.5-4 mg/ml) and control protein (1-2 mg/ml) were diluted proteinbuffer was dispensed on nitrocellulose membrane by Biodot low volumeprecision dispensing equipment. The nitrocellulose membrane is driedovernight under 10% humidity. A sample pad is dipped into a sample padbutter and is dried at 37° C. for overnight under 15% humidity. Asuspension of high density gold particle conjugated with a protein forthe detection of canine IgG is sprayed with pressure into slicedconjugate pad at a room temperature to obtain a high optical density.The conjugation pad was then dried for overnight at 25° C. under 10%humidity. The kit was assembled by putting nitrocellulose membrane on abacking pad, putting the sliced conjugation pad filled with goldparticle on backing pad which has to be overlapping with nitrocellulosemembrane in front area, putting the sample pad over conjugation pad,putting an adsorption pad overlapping with nitrocellulose membrane intail part, cutting the assembled backing pad by 4 mm wide and puttingthe cut assembled backing pad into a housing.

Since the above embodiments are described only for examples, the presentinvention is not limited to the above embodiments and variousmodifications or alterations can be easily made therefore by thoseskilled in the art without departing from the scope of the presentinvention.

What is claimed is:
 1. A lateral flow kit for quantitatively detectingan antibody of canine ECPKA in blood of a dog, comprising: a first solidphase having an immobilized purified recombinant canine PKA Cα proteinwherein the recombinant canine PKA Cα protein has an amino acid sequencecomprising SEQ ID NO. 005, a conjugate pad comprising a conjugatedcoloring agent with an optical density wherein the conjugated coloringagent is conjugated with a first binding protein, and a second solidphase comprising a second binding protein, wherein the conjugatedcoloring agent is configured to provide a first color intensity in thefirst solid phase and a second color intensity in the second solidphase, and wherein the first color intensity quantitatively depends onthe concentration of the antibody in the blood of the dog and the secondcolor intensity is independent of the concentration of the antibody 2.The lateral flow kit according to claim 1, wherein the optical densityof the conjugated coloring agent is between 5-30.
 3. The lateral flowkit according to claim 1, wherein the optical density of the conjugatedcoloring agent is between 10-20.
 4. The lateral flow kit according toclaim 1, wherein the lateral flow kit detects cancer in the dog with aspecificity of at least about 80% and a sensitivity of at least about80%.
 5. The lateral flow kit according to claim 1, wherein the firstcolor intensity provides the concentration of the antibody by comparingthe first color intensity with a set of correlating data between thefirst color intensity and the concentration of the antibody.
 6. Thelateral flow kit according claim 5, wherein a portion of the set ofcorrelating data can be expressed approximately by a linear equation,which allows determination of the concentration when the first colorintensity does not exactly match with any of the set of correlatingdata.
 7. The lateral flow kit according claim 1, wherein the firstbinding protein is selected from a group of streptavidin, biotin,protein A, anti-canine IgG Rat, anti-canine IgG Rabbit, anti-canine IgGgoat, anti-canine IgG sheep and a protein capable of binding to theantibody.
 8. The lateral flow kit according to claim 1, wherein thesecond binding protein is selected from a group of streptavidin, biotin,protein A, anti-rat IgG, anti-Rabbit IgG, anti-goat IgG, anti-sheep IgGand a protein capable of binding to IgG.
 9. The lateral flow kitaccording to claim 1, wherein the coloring agent is selected from agroup of gold, latex, gfp, fitc, and a UV active conjugating agent. 10.The lateral flow kit according to claim 1, further comprising a filterphase to filter blood cells.
 11. A method of determining a concentrationof an antibody of canine ECPKA in blood of a dog, comprising: (a)preparing a test sample from the blood of the dog for a lateral flowkit, (b) applying the test sample to the lateral flow kit, (c) allowingthe lateral flow kit to develop, (d) obtaining a digital information bytaking a digital picture of the developed lateral flow kit including thetest line, and (e) obtaining the concentration of the antibody whereinthe concentration is determined by comparing the digital informationwith a set of data correlating a digital value with a concentration ofthe antibody wherein the lateral flow kit comprises a first solid phasehaving an immobilized purified recombinant canine PKA Cα protein whereinthe recombinant canine PKA Cα protein has an amino acid sequencecomprising SEQ ID NO. 5, a conjugate pad comprising a conjugatedcoloring agent with an optical density wherein the conjugated coloringagent is conjugated with a first binding protein, and a second solidphase comprising a second binding protein, wherein the conjugatedcoloring agent is configured to provide a first color intensity in thefirst solid phase and a second color intensity in the second solid phasewherein the first color intensity quantitatively depends on theconcentration of the antibody in the blood of the dog and the secondcolor intensity is independent of the concentration of the antibody 12.The method according to claim 11, further comprising determining alikelihood that the dog has a cancer.
 13. The method according to claim11, further comprising sending the digital information is sent to anexternal server via a wireless communication.
 14. The method accordingto claim 11, wherein the obtaining the digital information is conductedusing a reader box comprising a wireless communication module, a cameramodule, a light source, and a slot designed to accommodate the lateralflow kit.
 15. The method according to claim 14 wherein the wirelesscommunication module operates based on a short range wirelesscommunication protocol.
 16. The method according to claim 11, whereinthe optical density is higher than
 5. 17. The method according to claim11, wherein the optical density is higher than
 10. 18. The methodaccording to claim 11 wherein the method detects cancer in the dog witha specificity of at least about 80% and a sensitivity of at least about80%.
 19. The method according to claim 11, wherein the concentration ofthe antibody is extrapolated using a linear equation with an R² valuehigher than 0.9.
 20. A method of determining a concentration of anantibody of ECPKA in blood of a mammal, comprising: (a) preparing a testsample from the blood of the mammal for a lateral flow kit comprising;(b) applying the test sample to the lateral flow kit; (c) allowing thelateral flow kit to develop; (d) obtaining a digital information bytaking a digital picture of the developed lateral flow kit; and (e)obtaining the concentration of the antibody wherein the concentration isdetermined by comparing the digital information of the test line with aset of data correlating a digital value obtained from the digitalpicture with a concentration of the antibody wherein the lateral flowkit comprises a first solid phase having an immobilized purifiedrecombinant mammal PKA Cα protein wherein the recombinant mammal PKA Cαprotein has an amino acid sequence, a conjugate pad comprising aconjugated coloring agent with an optical density of 5 or higher whereinthe conjugated coloring agent is conjugated with a first bindingprotein, and a second solid phase comprising a second binding protein,wherein the conjugated coloring agent is configured to provide a firstcolor intensity in the first solid phase and a second color intensity inthe second solid phase, and wherein the first color intensityquantitatively depends on the concentration of the antibody in the bloodof the dog and the second color intensity is independent of theconcentration of the antibody.